The second messenger cAMP is involved in regulating a variety of responses in virtually every cell in our bodies. One example is the cardiac myocyte, where beta-adrenergic receptors mediate sympathetic effects on electrical, mechanical, and metabolic activity through the production of cAMP. However, there are actually multiple receptors capable of regulating cAMP production in cardiac myocytes, just like many other cells. Even though various receptors may share this common second messenger, they do not all elicit the same functional responses. The common explanation is that cAMP production is compartmentalized, spatially limiting the extent of responses produced by certain receptors. The idea that activation of a receptor does not lead to uniform stimulation or inhibition of cAMP production throughout the cell might seem intuitively obvious, yet it is not fully understood how this is achieved. The common assumption is that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We will test the hypothesis that phosphodiesterase activity plays an important role in compartmentation, but not as a barrier to diffusion. We will also test the hypothesis that the cytoskeleton and cholesterol-dependent organization of the plasma membrane play essential roles in this process. The strength of this application lies in the innovative combination of methods that will be used to address these hypotheses. We will employ a systems biology approach that involves the use of multiple fluorescence resonance energy transfer (FRET) based biosensors to monitor cAMP activity in distinct subcellular compartments of live cells, together with quantitative computational modeling of compartmentalized cAMP signaling. The production of cAMP is an important means of eliciting beneficial changes in function of many cell types. However, cAMP-dependent signaling can also produce pathological responses under the right (or wrong) conditions. It has been hypothesized that the difference between the physiological and pathological effects may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with many different disease states. PUBLIC HEALTH RELEVANCE: The function of virtually every cell in our bodies is regulated by a myriad of neurotransmitters and hormones. Even though many of these compounds may produce responses involving a common signaling mechanism, the functional consequences are often quite different. The goal of this proposal is to understand how the cell orchestrates this complex behavior.